Clear cell renal cell carcinoma (ccRCC) is the most common renal malignancy worldwide. While metastatic ccRCC is refractory to conventional cytotoxic drugs and radiation, life-extending targeted therapies have emerged from an understanding of the molecular pathogenesis of this disease. In particular, over 80% of ccRCC cases exhibit constitutive activation of Hypoxia Inducible Factor (HIF)-dependent transcriptional responses, mediated by heterodimers of HIF- subunits (HIF-1 or HIF-22) and HIF-1. In the case of ccRCC, it is clear that HIF-2, but not HIF-1, is required for disease progression. While HIF-2 is not required for in vitro cell proliferation under oxygen, serum, and nutrient-replete conditions, xenograft mouse models have revealed that HIF-2 is essential for in vivo tumor growth, suggesting that HIF-2 mediates vital cellular stress responses within the in vivo tumor microenvironment. However, the downstream effectors of HIF-2's tumor-promoting activity remain incompletely understood. Using gene expression profiling, I have identified candidate HIF-2 regulated pathways in ccRCC by acutely suppressing HIF-2 activity by siRNA. These results indicate that HIF-2 regulates essential genes mediating mitophagy (NIX) and de novo lipogenesis (SREBF1, ACLY, ACACB, FASN, SCD1). NIX-dependent mitophagy plays a vital role in preventing oxidative stress by eliminating damaged mitochondria, the primary endogenous source of cellular reactive oxygen species (ROS). While HIF-2 has been demonstrated to promote resistance to ionizing radiation-induced oxidative stress and apoptosis, the precise mechanism is not clear. Thus, my first hypothesis (AIM1) is that HIF-2-dependent mitophagy is required to maintain redox homeostasis and support tumor cell metabolism. Additionally, constitutive de novo lipogenesis is needed for viability and growth in multiple malignancies, including breast and prostate carcinoma. Despite the observation that ccRCC are lipid laden, a phenotype lost upon HIF-2 suppression in xenograft tumor models, the role of de novo lipogenesis in ccRCC is unknown. Notably, HIF-2-dependent upregulation of TGF- expression is required for serum-independent in vitro proliferation-a condition that likely requires the synthesis of new fatty acids. Thus, my second hypothesis (AIM2) is that HIF-2-mediated TGF- expression activates SREBP1-dependent de novo lipogenesis to support tumor cell viability and proliferation. To test these hypotheses, I will utilize cell culture, a muine orthotopic renal tumor model, and primary patient samples to study the regulation of each process by HIF-2, evaluate the anti-tumor potential of suppressing these pathways (genetically and pharmacologically), and assess for activation of these processes in human tumors. These findings will provide novel insights into molecular pathogenesis of ccRCC, HIF biology, and potential anti-tumor therapies against ccRCC.